Venezuelan Equine Encephalitis virus (VEEV) is a life-threatening, NIH/NIAID category B human pathogen and a potential bioterrorism threat. Outbreaks of VEEV occur in Central America and have previously spread into the United States. The potentially devastating effects of the virus reemergence in the U.S. demand an effective vaccine to protect population. Currently, live attenuated TC-83 vaccine is used under IND protocol for vaccination of medical personnel at risk. The vaccine causes adverse effects, and efforts to develop an improved VEEV vaccine are underway. However, because vaccine development is a lengthy process and the supply of TC-83 vaccine is limited, the U.S. may soon experience a shortage of the VEEV vaccine. This can leave both the U.S. population and at-risk personnel unprotected. Furthermore, in the absence of vaccine, VEEV may require re-classification as a BSL4 Select Agent. In Phase I SBIR, we developed a new technology for vaccination against VEEV and, potentially, other viral diseases. The proposed iDNA vaccination technology represents a unique combination of conventional DNA immunization with the high efficacy of live attenuated vaccines. The key feature of this technology is that live attenuated virus is launched in vivo from iDNA plasmid carrying a molecular clone of VEEV vaccine with enhanced safety and immunogenic features. In Phase I SBIR studies we have shown that injection in vivo of the prototype iDNA derived from the TC-83 vaccine has successfully launched live attenuated vaccine in mice. In this Phase II SBIR we propose advanced preclinical evaluation of iDNA VEEV vaccine based on the rational engineering of TC-83 clones and iDNA immunization technology. In Sp. Aim 1, we propose (i) optimization of iDNA vaccination in vivo including iDNA formulation and the route of administration with, and without, electroporation, and (ii) dose escalation study to determine the minimal amount of iDNA sufficient to launch the vaccine virus and to induce protection in BALB/c mice. The iDNA will be formulated to minimize the need for electroporation and cold chain. In summary, the goal of Sp. Aim 1 is the development of patient- and doctor-friendly procedure for iDNA vaccination. In Sp. Aim 2, in collaboration with the University of Louisville, KY (UofL) we propose evaluation of safety, immunogenicity and efficacy of experimental VEEV iDNA vaccines in mice, rabbits, as well as in rhesus non-human primates (NHP), which represent the best model for human VEEV infection. As a control, the standard TC-83 vaccine will be used. Following these studies, the lead VEEV iDNA vaccine will be selected for the cGMP production during Sp. Aim 3. In addition, we propose a pre-IND meeting with the FDA to seek input on the design of (i) GLP toxicology study and (ii) Phase I clinical trial. Our preliminary data suggest that the rational vaccine design and iDNA technology can provide a revolutionary solution for VEEV vaccine by improving safety, genetic stability, and immunogenicity, and by eliminating many costly steps of the conventional manufacturing process. Essentially, live attenuated vaccine will be manufactured within the immunized individuals. This technology also utilizes many advantages of DNA vaccines (genetic homogeneity and stability, low cost of manufacturing, storage, and transportation, no cold chain) and, more importantly, enhances immunogenicity. As any recombinant DNA, the iDNA activates cGAS-cGAMP-STING-dependent signaling pathways resulting in robust production of cyto- and chemokines, which induce strong priming effects and stimulate acquired virus-specific immune responses. The final iDNA VEEV vaccine will represent a novel class of vaccines combining the advantages of DNA and live attenuated vaccines. The iDNA technology can be easily adapted for the development of other vaccines including live attenuated vaccines for WEEV, EEEV, other alphaviruses, and flaviviruses. If successful, this technology can potentially transform the field of live attenuated vaccines for many viral diseases.